Proteomics reveal biomarkers for diagnosis, disease activity and long-term disability outcomes in multiple sclerosis

Sensitive and reliable protein biomarkers are needed to predict disease trajectory and personalize treatment strategies for multiple sclerosis (MS). Here, we use the highly sensitive proximity-extension assay combined with next-generation sequencing (Olink Explore) to quantify 1463 proteins in cerebrospinal fluid (CSF) and plasma from 143 people with early-stage MS and 43 healthy controls. With longitudinally followed discovery and replication cohorts, we identify CSF proteins that consistently predicted both short- and long-term disease progression. Lower levels of neurofilament light chain (NfL) in CSF is superior in predicting the absence of disease activity two years after sampling (replication AUC = 0.77) compared to all other tested proteins. Importantly, we also identify a combination of 11 CSF proteins (CXCL13, LTA, FCN2, ICAM3, LY9, SLAMF7, TYMP, CHI3L1, FYB1, TNFRSF1B and NfL) that predict the severity of disability worsening according to the normalized age-related MS severity score (replication AUC = 0.90). The identification of these proteins may help elucidate pathogenetic processes and might aid decisions on treatment strategies for persons with MS.


List of Figures:
A group (n = 21) of persons with MS (pwMS; replication cohort group A) had higher expression of several protein markers for delayed sampling handling (TNFSF14, SIRT2, CASP8, AXIN1) in plasma.The figure shows the expression of the top six most affected proteins, also measured on the Olink panel, by sampling handling variability from Table 1 in [1].The pwMS from both the discovery and the replication cohort were divided into two groups (A and B), which in both cohorts represents samples from an old collection (A) and from a new collection (B).The remaining groups of pwMS (discovery cohort group A and B, and replication cohort group B) and the healthy controls (HC) did not show any clear effect of sampling or handling variability.

Figure S 2. A principal component analysis of the plasma samples from persons with MS (pwMS).
The plasma samples from one group (replication cohort group A, n = 21 samples) of pwMS are to a high extent clustering separately compared to the samples from the remaining groups of pwMS (discovery cohort group A and B, and replication cohort group B).The pwMS from both the discovery and the replication cohort were divided into two groups (A and B), which in both cohorts represents samples from an old collection (A) and from a new collection (B).

Figure S 10. Portions of samples below the limit of detection (LOD) in persons with MS (pwMS) and healthy controls (HC).
The portions of samples below the LOD in the different groups, pwMS and HC, in the discovery cohort and the replication cohort, separately.The 52 differentially expressed proteins in the discovery cohort are shown.

Figure S 12. Portion of samples below limit of detection (LOD) in persons with MS (pwMS) and healthy controls (HC): comparison between discovery and replication cohorts.
The portion of samples below the LOD in the different groups, pwMS and HC, in the discovery cohort and the replication cohort, separately.The figure shows the proteins removed during the pre-processing of the protein data, based on more then 75% of the samples being below LOD, but which had less than 75% of the NPX values below the LOD in either samples from pwMS or HC in the discovery cohort.
A document from the Swedish MS Association that categorizes DMTs into low, medium or high efficacy (only available in Swedish).On pages 3 and 4, the treatment classifications are listed as low-(lägre effekt), medium-(måttlig effekt) or high-(hög effekt) efficacy.We included the low effect drugs as first-line treatment and the high effect drugs as second-line treatment in our study.We classified the medium effect drugs as either first-line or secondline based on the literature. https://www.mssallskapet.se/wp-content/uploads/2023/04/SMSS-Nationella-riktlinjer-forvard-vid-multipel-skleros-230412.pdf

Figure S 3 .
Figure S 3. Heatmap of differentially expressed proteins in cerebrospinal fluid.A clustered heatmap showing the protein expression (NPX) of the differentially expressed proteins (false discovery rate < 0.05) either in the discovery cohort or the replication cohort in the cerebrospinal fluid.Each column shows the expression of one sample.At the top of the heatmap it is shown which groups a sample is part of (person with MS or healthy control (HC) and discovery cohort or replication cohort), sex (female or male), and age at baseline (16 -64 years) of each person.

Figure S 5 .
Figure S 5. Heatmap of differentially expressed proteins in cerebrospinal fluid (CSF) and plasma in persons with MS vs. healthy controls.A heatmap showing the log2 fold-change (FC) values of the differentially expressed proteins (false discovery rate (FDR) < 0.05) either in the discovery cohort or the replication cohort in the CSF, comparing persons with MS to healthy controls.The log2FC values in the CSF are compared to the log2FC values in plasma.The differential expression analysis was performed using a two-sided linear model t-test (Limma analysis).

Figure S 6 .
Figure S 6.The correlation between cerebrospinal fluid samples and plasma samples.The correlation, assessed with Pearson's correlation coefficient (PCC), for each of the 52 differentially expressed proteins in the discovery cohort are shown.The significance of the PCCs was assessed with t-statistics (two-sided): * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure S 7 .
Figure S 7. Correlations between treatment duration index and protein expression.The correlations, assessed with Spearman's correlation coefficient (SCC), between treatment duration index and the expression of each of the 52 differentially expressed proteins in the discovery cohort.For the normalized age-related MS score (nARMSS) models the treatment duration index is based on the full follow-up time from baseline and for the disease activity models the treatment duration index is based on two years from baseline.The significance of the SCCs was assessed with t-statistics (two-sided): * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure S 8 .
Figure S 8. Individual plots: expanded disability status scale (EDSS) scores of persons with MS.Individual plots for each person with MS showing the person's EDSS scores (blue dots) during the follow-up years after baseline sampling (discovery cohort: n = 71 samples; replication cohort: n = 33 samples).Plots are shown for persons with at least three years follow-up.Documented relapses are shown with a red vertical line.On top of each plot the person's age at baseline sampling and the calculated normalized age-related MS scores (nARMSS) are stated.

Figure
Figure S 8.

Figure S 9 .
Figure S 9. Normalized age-related MS score (nARMSS) distribution for persons with MS (pwMS).Histograms showing the distribution of normalized age-related MS scores (nARMSS) for pwMS in the discovery cohort (n = 71 samples; left) and the replication cohort (n = 33 samples; right).

Figure S 13 .
Figure S 13.Singular value decomposition analysis: uncorrected and corrected NPX values in cerebrospinal fluid (CSF) and plasma samples.Singular value decomposition analysis of the uncorrected NPX values (left) and the corrected NPX values (right) in both the CSF ( top) and plasma (bottom) samples.The NPX values were corrected in two steps: 1) control corrected: NPX values were adjusted so that controls in the discovery cohort and the replication cohort had the same standard deviation and mean, 2) ComBat corrected.The categories investigated were group (person with MS or healthy control), sex (female or male), age at baseline, and site (sampled at Linköping university hospital or Karolinska university hospital).The significance of the correlation of each category with each principal component (PC) is shown in the plot.

Table S 1
. Known MS biomarkers with supporting references.

Table S 2
. Performance of the 52 differentially expressed proteins in cerebrospinal fluid in the discovery cohort for predicting MS diagnosis.Area under the curve (AUC) scores in discovery and replication cohorts are shown.The significance of the AUC scores were assessed with a two-sided Mann-Whitney U test.Performance of the 52 differentially expressed proteins in cerebrospinal fluid in the discovery cohort for predicting disease activity.Area under the curve (AUC) scores in discovery and replication cohorts are shown.The significance of the AUC scores were assessed with a two-sided Mann-Whitney U test.

Table S 4
. A linear regression model consisting of 11 cerebrospinal fluid (CSF) proteins could predict normalized age-related MS score (nARMSS).The coefficients of the combined model are compared to the predictive power of the individual proteins.For the individual protein models, the coefficient and performance of each model is stated.The performance is assessed with Spearman's correlation coefficient (SCC) and Lin's concordance correlation coefficient (CCC) between the true nARMSS and predicted nARMSS.Age was included as a predictor in the models for all individual proteins.Last, the expression in CSF for four out of the 11 proteins were correlating with the expression in plasma, assessed with Pearson's

Table S 6
. References for proteins in the MS network that were not annotated with a Gene Ontology term.

Table S 7
. References for the drug classification into first-line (low-efficacy) and second-line (high-efficacy).